KR20180108920A - Method and apparatus for the analysis and identification of molecules - Google Patents

Abstract

An apparatus and method for performing analysis and identification of molecules are provided. In one embodiment, the portable molecular analyzer comprises a sample input / output connection for receiving a sample, a nanopore-based sequencing chip for performing analysis on a sample in substantially real time, and an output interface for outputting the results of the analysis .

Description

[0001] METHOD AND APPARATUS FOR THE ANALYSIS AND IDENTIFICATION OF MOLECULES [0002]

The present invention relates generally to the analysis and identification of molecules, and in particular to providing a real-time, portable, nanopore-based molecular analysis apparatus.

There are nucleic acids, DNA (deoxyribonucleic acid), and / or RNA (ribose nucleic acid) and have unique sequences in all living organisms. This lends itself naturally as a definitive identification for a variety of bio-agents. Thus, analysis of nucleic acids, DNA and / or RNA, which is widely referred to herein as genomic analysis, is very useful for studying living organisms. However, there is a need for a method and a method for sequencing by ligation, such as microarray, pyrosequencing, sequencing by synthesis by synthesis, and sequencing by ligation, The nucleic acid sequencing technologies are very limited in various respects. For example, some or all of these techniques are not capable of real-time analysis, require long sample nucleic acid amplification processes and protocols (such as polymerase chain reactions), and typically require the analysis of small fragments of the sample nucleic acid (Some of them use expensive chemical reagents), high false-positive error rates (which take about a few days to several weeks to run), high operating costs , And can not carry.

Due to the above limitations of current nucleic acid sequencing techniques, people working in the field, such as medical professionals, security personnel, scientists, etc., can perform genome analysis locally in the field none. Rather, field workers must collect and transport samples to specific laboratories that will be analyzed for days or weeks to identify nucleic acids present in the sample. During a particularly epidemic epidemic, such as foot-and-mouth disease in the United States, Severe Acute Respiratory Syndrome (SARS) outbreaks in Asia, and the recent outbreak of H1N1 flu (also commonly known as swine flu) in Mexico and the United States, Can hardly meet today's demands for genome analysis. Using current nucleic acid sequencing techniques, it is difficult, although not impossible, to formulate information-based decisions quickly, where authorities can have enormous safety and economic impacts on society.

To cope with the lack of such nucleic acid sequencing techniques, scientists have developed a variety of nanopore-based sequencing technologies. Recently Professor Hagan Bayley and his colleagues at Oxford University have used the α-haemolysin in a bio-nanopore experiment to develop a long reading with 99.8% accuracy long read). Based on the established detection rate, an array of 256 x 256 nanopores is generally sufficient to analyze the human genome in its entirety within about 30 minutes. If you can successfully realize a bio-nanopore array, this is a critical time triumph. However, one drawback to bio-nanopores is the relatively short life span of proteins and enzymes used to form bio-nanopores, typically from hours to days.

Solid state nanopores are a more robust alternative to bio-nanopores because there is no bio-reagent contained in the constitution of solid nanopores. However, conventional lithography techniques employed in the semiconductor industry are not able to define the 2 nm shape size required by solid nanopore-based sequencing techniques. Thus, further manufacturing techniques, such as electron / ion milling, are in turn being used to continuously engrave nanopores. However, these techniques can not be expected to produce a 256 x 256 array with reasonable cost and reasonable production time.

An object of the present invention is to provide a real-time, portable, and nanopore-based molecular analysis apparatus.

The present invention provides a real-time, portable, nanopore-based molecular analysis device.

Figure 1 shows one embodiment of a nanopore-based sequencer and related nanopore-based sequencing biochip.
Figure 2a shows one embodiment of the translocation process of a molecule during detection and analysis of nanopore-based nucleic acid sequence determinations.
Figure 2b shows a corresponding exemplary electrical readout of the nucleic acid sequence as a comparison with the background signal of the empty pore.
Figure 3 is a side view and top view of one embodiment of an Einzel lens used in nanopantography for both etch and deposition.
4A to 4E show one embodiment of a subtractive method for manufacturing a nanopore and / or a nanopore array.
Figures 5A-5I illustrate one embodiment of an additive method for making nanopores and / or nanopore arrays.
Figure 6 shows one embodiment of nanorring and one embodiment of a nanoslit.
Figure 7 shows one embodiment of a bonded nanopore array wafer and an integrated circuit wafer for forming a bottom cavity of a measurement chamber.
Figure 8 shows one embodiment of a bonded top wafer and a composite wafer to form the top cavity of the measurement chamber.
Figure 9 shows one embodiment of a current sensing circuit and a voltage biasing scheme that can operate with a nanopore-based sequencer.
10 (a) shows one embodiment of a nanopore-based nucleotide probe.
Figure 10B shows an embodiment of multiple measurement chambers.
Figure 11 shows a cross-sectional view of one embodiment of nanopore-based nucleation along a selected path from a sample inlet to a sample outlet, according to a microfluidic channel and a nanofluidic channel, through a measurement chamber .
Figure 12 shows one embodiment of a trilayer biochip structure with buried electrodes.
13A shows an embodiment of a biasing and sensing structure for nanopore detection.
Figure 13b shows an embodiment of a planar electrode implementation.
Figure 13c shows a top view of one embodiment of sensing electrodes and nanoslit in a planar electrode implementation.
14 shows a high-level hardware architecture of one embodiment of a nanopore-based nucleotide probe.
Figure 15 shows the high-level structure of software and associated hardware for genome analysis software and operating systems in one embodiment of nanopore-based nucleotide sequencing.

In the following description, for purposes of explanation, numerous specific details are set forth such as examples of specific elements, devices, methods, and so forth in order to provide a thorough understanding of embodiments of the present invention. However, it will be apparent to those skilled in the art that these specific details need not be employed in an actual embodiment of the present invention. In other instances, well-known materials or methods are not described in detail in order to avoid unnecessarily obscuring embodiments of the present invention.

Nucleic acids, amino acids, peptides, proteins and polymers, and carbon nanotubes, silicon nanorods, and coated / uncoated Various embodiments of apparatus and methods for performing analysis and identification of molecules, such as nanometer sized particles, such as coated / uncoated gold nanoparticles, are described below. It should be noted that the following discussion, to illustrate the concept, focuses on one example of molecular analysis, i.e. genome analysis. One of the prior art is readily recognized that the techniques disclosed herein are generally applicable for analyzing and identifying molecules. In one embodiment, the nanopore-based sequencer is a portable genomic analysis system. FIG. 1 shows an embodiment of a portable nanopore-based nucleotide probe 110 and a related nanopore-based nucleotide sequencing biochip 120. As shown in FIG. 2A, during detection and analysis, the molecules in the sample under test are electrophoretically moved from the solution through a nano-scale pore (also referred to as a nanopore) (electrophoretically) driven. In some embodiments, the size of the nanopore is about 2 nm. It should be noted that the size of the nanopore may vary in other embodiments. For example, in some embodiments, the nanopores are of the same fixed size. In some other embodiments, the nanopores are of different sizes. Moreover, the shape of the nanopore may also vary in the same or other embodiments, such as circles, ovals, elongated slits, and the like. Various electrical characteristics, such as current pulses of different amplitudes and intervals, are observed to identify the molecules, as shown in Figure 2B, subject to the relative size and traveling speed of the molecules in the space defined within the nanopore Can be used. As a result, direct reading of the nucleotide sequence can be achieved without destroying the molecule under test. That is, the measurement can be made against the nucleotide sequence while maintaining the nucleotide sequence integrity.

Multiple fabrication techniques can be used to fabricate large quantities of nanopore arrays, including in some embodiments, arrays of about 2 nm pores, without fundamental limitations. The details of some exemplary fabrication techniques are described in detail below to illustrate the concepts. One of the prior art is recognized that other comparable manufacturing techniques or variations for these techniques can be employed to fabricate the nanopore array. By integrating into the network of micro- / nano-fluidic channels, nanopore-based nucleotide sequencing can precisely decode the genome without human intervention at unprecedented rates.

In addition to small form-factor and rate in genome analysis, some embodiments of nanopore-based nucleotide sequencing provide the following additional advantages. One advantage is the future production competitiveness of ready-made production for any mutation in bio-agents. This is possible because nanopore-based nucleotide sequencing is a direct reading technique in which the results do not require prior knowledge of the genome under test. Furthermore, during the entire analysis process, the nanopore is always surrounded within the nanopore-based nucleotide sequence determination biochip and is not exposed to a predetermined undesired foreign substance, so that sterility and cleanliness are always guaranteed for the nanopore Thus, some embodiments of nanopore-based nucleotide sequestration can operate in extreme and filthy environments.

As a pocketable device, some embodiments of nanofoer-based nucleotide heating can accelerate progress in many different industries and sciences. For example, in commercial as well as research and development, some embodiments of nanopore-based nucleotide probes may be useful in basic studies, pharmacogenomics, bacterial diagnostics, pathogen detection, agriculture, the food industry, biofuels, and the like. As another example, some embodiments of nanopore-based nucleotide probes may be useful in fast DNA forensics, port-of-entry bio-screening, and the like.

Nanopore arrays for nucleotide-based sequencing

On the basis of the resistive-pulse technique of the Coulter Counter in the 1970s, DeBlois and colleagues found that single- and double- Has successfully demonstrated the use of single submicron diameter pores. Subsequently, Deamer proposed the idea of using nanometer sized pores for gene sequencing. He and his colleagues found that single-stranded DNA (ssDNA) and RNA molecules can be driven through pore-forming proteins and detected by their effects on ionic currents through these nanopores . Given the recently proven high nucleotide sequencing rates, advances in nanopore-based sequencing are inexpensive to produce large arrays of nanopores for rapid genome analysis and lack a parallel-write fabrication process Lt; / RTI > Most conventional lithography methods, electron milling, ion milling, and silicon etch back are not practical tools for fabricating the nanopore arrays required for real-time genome analysis. Until recently, Donnelly and colleagues at the University of Houston have developed several embodiments of nanopantography capable of mass-producing 2 nm nanopore arrays without much limitation. According to their simulation results, nanotransfer may define holes or dots with sizes as small as 1 nm. By integrating the technology of micro- / nano-fluid, nanotransformation opens up the possibility of achieving close real-time or real-time genome analysis systems.

In nano transfer, a broad, collimated, monoenergetic ion beam is formed on a conductive substrate, for example, a submicron diameter antireflection lens fabricated on a doped silicon (Si) diameter electrostatic lenses (also referred to as Einzel lenses, as shown in Fig. 3). By applying an appropriate voltage to the lens electrode, the "beamlets" entering the lens are focused on spots that can be 100 times smaller than the diameter of the lens. This means that a 1 nm shape can be defined by a 100 nm lens, which can be processed by photolithography techniques used in current semiconductor processing. In addition, each lens writes its own nanometer geometry on the substrate, and thus the nanotransfer method is a parallel-write process and is very different from the sequential writing of a focused electron beam or ion beam . Because the lens array is part of the substrate, the method is substantially safe for misalignment caused by vibration or thermal expansion. The size of the nanometer geometry can be controlled by the size of a predefined lens, which diameters can be the same or different, for example arrays of the same or different nanometer shapes can be processed substantially simultaneously. Using Ar + beams in the presence of Cl ₂ gas, Si holes etched to a 10 nm diameter, 100 nm depth were demonstrated. The etching may be caused only in the holes to which the voltage is applied to the top metal so that the ion beam is focused. The remainder of the hole will not have a voltage applied to the top metal layer, the beamlets will not be focused, and the current density will be too small to cause any suitable etching.

There are two methods, namely subtractive method and additive method, for producing nanopores for genome analysis according to nano transfer method. One embodiment of the direct etch method is discussed first below, followed by a discussion of one embodiment of the indirect etch method.

A. Nanopores and / or nanopore arrays

 One embodiment of a subtraction method for manufacturing

4A to 4E show one embodiment of a subtraction method for producing a nanopore and / or a nanopore array. Referring to FIG. 4A, a nitride 420 is deposited on a Si (100) wafer 410. 4B, a bottom conducting material 422 doped with silicon or metal is deposited, and a dielectric spacer 424 and a top metal layer 426 are deposited. Next, a plurality of Einzel lenses is defined. 4C, a nanopantographic etch is performed on the conductive material 422 to define the nanometer hole 430, which is a hardmask for nanopore etching in the nitride layer 420, . In Fig. 4D, the Einzel lens is removed. The nanopore 430 is then coated with oxide 435 for protection. 4E, a bottom cavity 440 of the measurement chamber is formed on the back surface of the silicon substrate 410 by chemical mechanical polishing (CMP), lithographic technique, and potassium hydroxide (KOH) etch . The oxide layer 435 is then removed to expose the nanopore 430.

B. Nano-pores and / or nanopore arrays

 One embodiment of the addition method for manufacturing

5A to 5I show one embodiment of the addition method for manufacturing the nanopore and / or nanopore array. Referring to FIG. 5A, a nitride 520 is deposited on a Si (100) wafer 510. 5B, a bottom conductive material 522, a dielectric spacer 524 and an upper metal layer 526 doped with silicon or metal are deposited. Next, an Einzel lens is defined. In FIG. 5C, nanorods (530) for nanorod or nanotube growth are deposited. In Fig. 5D, nanorods or nanotubes 535 are grown. In Fig. 5E, an oxide 540 is deposited. In Fig. 5F, the nanorods or nanotubes 535 are removed. The remaining oxide nanometer holes 550 are used as a hard mask for the conductive and nitride layers. 5G, a pattern is transferred from the oxide layer 540 to the conductive layer 522 and then to the nitride layer 520. In FIG. 5H, the Einzel lens is removed and removal of the oxide layer 540 is continued. The nanopore is coated with oxide 552 for protection. Finally, in Figure 5i, a bottom cavity 560 of the measurement chamber is formed by CMP, lithographic techniques and KOH etching on the backside of the silicon substrate 510. The oxide layer 552 is then removed to expose the nanopore 565.

Figure 6 illustrates one embodiment of nanoslit and one embodiment of nanoring that can be used in some embodiments of the invention. The extended nano-sized nano-slits 610 and the nano-rings 620 are formed by a subtractive method according to an Einzel lens (discussed above) having openings in the form of rectangular and semicircular, respectively, ≪ / RTI > It will be apparent to those skilled in the art, based on the disclosure provided herein, that certain two-dimensional shapes can be defined using similar patterning techniques. In some embodiments, the non-circular patterning method solves at least three major technical problems encountered by conventional methods of tilting the wafer stage, since the wafer stage is substantially stationary during the entire process. do. First, precise control of the speed and tilt angle of the wafer stage is not necessary. Second, we generally overcome the line-width non-uniformity and line-broadening effects induced by tilting the wafer with respect to the incoming beam of ions . Third, other shapes and sizes of patterns that are defined at approximately the same time are allowed.

Nanopore-based sequencing biochip

After the nanopore array is formed according to a subtraction or addition method, as shown in FIG. 7, the nanopore array wafer 750 is pre-fabricated with pre-fabricated integrated circuits 730 and microfluidic channels 730 (FIG. RTI ID = 0.0 > microfluidics < / RTI > channels. This completes the formation of the bottom cavity of the measurement chamber. The nucleic acid sample can be extracted outside the biochip through a microfluidic channel on the bottom wafer, if desired.

Likewise, in some embodiments, the top cavity of the measurement chamber may be integrated onto a composite wafer, including the bonded nanopore wafer 840 and the bottom wafer 850, / RTI > and / or fluid channel (830). This three-layer wafer configuration 800 is shown in FIG. One embodiment of voltage biasing structure 910 and current sensing circuit 920 is shown in FIG. According to the appropriate fluid I / O connections, such as having a minimal dead-volume, the three-layer composite wafer 800 may then be subjected to wire bonding or bell grid wire And mounted on a supporting frame for bell grid wire bonding. Typical packaging techniques, such as epoxy encapsulation and ceramic packaging, can be used to encapsulate the entire assembly to form a nanopore-based sequencing biochip.

On the other hand, integrated circuits, such as those associated with sensing and biasing, can be fabricated on a nanopore wafer 840, which can be bonded to a blank substrate instead of another wafer with integrated circuitry.

10A and 11, the microfluidic channel 1010 and the nanofluid channel 1010, which lead to the measurement chamber 1030, have two or more features embedded on the top wafer 810. In some embodiments, There are sample guiding electrodes 1015 along the nanofluidic channel 1013. [ In order to further illustrate the flow of the sample through the nanopore-based sequencing biochip, one example is discussed in detail below.

The buffer intake 1025 and the buffer outlet 1027 are connected to the network of the microfluidic channel 1010, the nanofluidic channel 1013 and the measurement chamber 1030 prior to accepting the sample for detection. It is wet and pre-fill. During the detection, the fluid flow in the microfluidic channel is controlled by the on-chip or off-chip micropumps and microvalves to the flow rate of the buffer inlet and outlet Lt; / RTI >

In one embodiment, the phosphate-deoxyribose backbone of a single strand nucleic acid molecule is filled with a negative charge for each base segment, There are two negative charges at the 5'-end of the molecule. Positive voltage pulsating along the sample guiding electrode chain 1015 from the receptive reservoir 1020 through the sample inlet connector 1023 to the predetermined measuring chamber 1030 can be obtained from the receiving reservoir 1020 Nucleic acid molecules can be extracted and molecules transferred to the preassigned measurement chamber 1030. [ Similarly, the sample guide electrode 1017 is also embedded on the bottom wafer along the microfluidic channel 1010 for sampling the sample out of the nanopore-based sequencing biochip in a similar manner.

Using a similar structure of the sample guide electrode along the network of fluid channels, the number of measurement chambers 1040 can be extended to one or more. An example of a measurement chamber arranged in a tree structure is shown in Fig. 10B. In addition to the ability to perform multiple independent assays, the order of the DNA fragments is pre-assigned for each measurement chamber in some embodiments as they are extracted from the sample acceptance reservoir 1020. As shown in FIG. 10B, the measurement chamber 1040 is labeled with a two-digit number. The first digit indicates the branch number and the second digit indicates the position of the measurement chamber in the branch. The order of assignment of the measurement chambers 1040 may simply be ascending, for example, a chamber with the lowest number may be used first, followed by a chamber with the next higher number. In this way, all measurement chambers of the branch can be used before moving to the next branch. On the other hand, the samples can be assigned to chambers having the lowest number in each branch as the lowest branch number is used first, for example 11, 21, 31, 41, 12, 22, 32, 42, etc. in the current sample. According to this allocation approach, each branch of the measuring chamber with the greatest distance from the central microfluidic channel can be allocated first, and then one of each branch can be assigned. This approach can reduce the interference of the electrical signal of the sample guide electrode in the central microfluidic channel on the measured signal. When the entire measurement is complete, the base sequence of the entire DNA sample can be systematically assembled according to the extraction order of the fragments. This is because the hybridization process randomizes the original nucleotide sequence of the sample and causes computational intensive and error-prone post-detection analysis to reverse the appropriate nucleotide sequence it is possible to omit the detection analysis after the time required by other conventional base sequence determination techniques, such as microarrays, which are required to back up, are consumed. Moreover, since the extraction sequence of the fragments is recorded, the fragments can be recombined to form the original DNA sample. For other conventional sequencing techniques, the original sample is typically destroyed and can not be retrieved for future use

Referring back to FIG. 10a, the nanofluidic channel 1013 formed by wafer bonding of the upper and composite wafers can serve as a filter, a sample flow rate controller, as well as a molecular stretcher. In some embodiments, the nanofluidic channel functions as a filter by turning on and off the upper electrode on the upper wafer, and is selectively attractable from the sample from the microfluidic channel. In some embodiments, the nanofluidic channel functions as a sample flow rate controller by adjusting the voltage at the top and bottom electrodes, and can control the flow rate of the sample through the nanofluidic channel. As shown in FIG. 11, since single-stranded nucleic acid molecule 1101 can be stretched outward from a natural curl up state when passing through nanofluidic channel 1013, 1013) can also function as a molecular stretcher.

In some embodiments, the velocity control characteristics of the nanofluidic channel are exploited to allow more accurate analysis of the molecule. 12, the sensing electrode 1205 is embedded in the nanopore 1225, which is an integral part of the micro / nano-channel network in controlling the flow of molecules through the nanopore 1225 . In addition to the nanofluidic channel, the applied DC voltage is designed to finely adjust the speed and direction of the molecules when translocating through the nanopore 1225. [ By alternating the magnitude of the DC biasing voltage applied to the top and bottom drive electrodes 1230 and 1235 and the embedded sensing electrode 1205, the accuracy in identifying the molecules can be increased by eliminating statistical errors The molecules under test can be pulled back and forth several times through the nanopore 1225 for repeated analysis, for example, a false-positive error rate can be reduced.

Unlike some conventional approaches, where the sensing electrodes are integrated in a nanopore, only a few nanoseconds are taken for each base in the DNA to travel through the nanopore. This transit time is too short for any meaningful measurement. In view of this drawback, other conventional approaches have been developed to reduce the rate of molecular migration through nanopores. One conventional approach suggests a voltage trapping scheme to control the rate of molecules by embedding extra electrodes into the nanopore. The proposed voltage trapping configuration is difficult to implement because it requires four or more electrically isolated electrodes that are electrically insulated from each other by sandwiching the dielectric material between the conductive electrodes stacked on top of each other. The required 2-3 nm nanopores formed on this multilayer film can have an aspect ratio of 30: 1 or higher, and to achieve with current integrated circuit fabrication techniques, although not impossible, it's difficult.

13A, the applied AC sensing voltage 1310 is for interrogating the molecules while moving through the nanopore 1325. As shown in FIG. As a result, various sensing mechanisms can be employed to identify molecules such as resistance change, capacitance change, phase change and / or tunneling current. Due to the proximity of the molecules under test and the embedded sensing electrodes 1305, the signal-to-noise ratio can be improved.

The exemplary sample transfer and filtering mechanism described above serves as an example to illustrate how an array of nanopore measurement chambers can be implemented. Those skilled in the art will appreciate that variations on the above-described delivery and filtering mechanisms may be employed in other embodiments. Moreover, arrays of pores having different sizes can be realized using the methods shown. An array of bio-nanopores can also be realized, along with protein pores such as alpha -hemolysin, and an array of such solid nanopores. In some embodiments, both sensing electrodes may be located on the same conductive layer instead of the stacked electrodes on the other conductive layer. 13B shows an embodiment of a planar electrode implementation. In this planar electrode implementation, the positive sensing electrodes 1307 are located on the same conductive layer with the nanoslots 1327 defined between the sensing electrodes 1307. A top view of the sensing electrode 1307 and the nano slit 1327 is shown in Fig. 13C. As described above, both nanopores and nanoslots can be defined using nanotransfer method.

The ability to perform molecular detection in substantially real time, the ability to perform single molecule detection without pre-detection sample amplification, the ability to perform multiple and substantially simultaneous detection, the ability to perform multi- the ability to identify samples without computational intensive post-detection analysis, and / or the ability to retain samples after detection for future use, can be used, for example, for single nucleotide polymorphism (single-nucleotide polymorphism) fast and accurate genome analysis in some embodiments that recognize nucleotide polymorphism.

Nanopore-based sequencer

The nanopore-based nucleotide sequence provides a portable genome detection and analysis system. In some embodiments, the nanopore-based nucleotide sequence includes 12 major components: hardware and software. Some embodiments of high level architectures and subunits are discussed below.

A. Hardware System

In some embodiments, the nanopore-based nucleotide sequencing hardware system includes two major units: computation, communication and control units and nanofoer-based sequencing biochip interface units and various modules. One embodiment of a high level structure is shown in FIG. Details of the various components are described below.

I. Computation, Communication and Control Unit (Computing, Communication, and Control Unit)

In one embodiment, the hardware system 1400 includes a portable computing system 1410 having a display device. This can be implemented using a tablet, a notebook, a netbook, an all-in-one desktop computer, a smart phone, a personal digital assistant (PDA), or a predetermined pocket calculator. A central unit for executing an operating system (OS), executing data analysis software, storing data, controlling the operation of the nanopore-based nucleotide sequence determination biochip, and collecting data from the nanopore-based nucleotide sequence determination biochip .

In one embodiment, the hardware system 1400 further comprises a network communication module 1420. Network communication module 1420 includes one or more WiFi, WiMAX, Ethernet, Bluetooth, telephone capability, satellite link, and Global Positioning System (GPS). The network communication module 1420 may communicate with a central computing system for data sharing, program update, data analysis, etc., communication with other computing devices where data can be shared and data analysis can be performed in parallel on multiple computing devices , Data sharing, program updates, and the like, and other Bluetooth capable devices (e.g., cellular phones, printers, etc.) that transmit and receive GPS signals.

In one embodiment, the hardware system 1400 further includes an input device 1430. The input device 1430 may include one or more keyboards, a touch screen, a mouse, a trackball, an infrared (IR) pointing device, and a voice recognition device. The input device 1430 functions as a human interface for a command entry and a data entry.

In one embodiment, the hardware system 1400 includes an input / output (I / O) port, which may include a flash memory card interface, an IEEE 1394 interface, and a Universal Serial Bus (USB) (1440). The I / O port 1440 functions as a serial interface with other electronic devices, a secondary data storage interface, and measurement data I / O for a nanopore-based sequencing biochip.

II. The nanopore-based sequencing biochip interface unit (Nanopore-based Sequencing Biochip Interface Unit)

In some embodiments, the nanopore-based sequencing (nSeq) biochip interface unit 1450 includes an nSeq electronics module 1460, a fluid control module 1470, a chemical reservoir and a fluid I / O connection module 1480, and the nSeq fluid control and sample I / O connection module 1490. The nSeq electronics module 1460 controls the distribution of the nucleic acid modules, the flow rate control of the nanofluidic channels, the collection measurement data, and the output data for the computation, communication, and control units.

In some embodiments, the fluid control module 1470 may include fluid flow between the nSeq biochip and the chemical reservoir via buffer inlet / outlet connectors, on-chip or off- And controls the use of off-chip micropumps and microvalves. The chemical reservoir and fluid I / O connection module 1480 may provide chemical to the nSeq biochip, if necessary, and also may contain chemical and / or bio-waste storage ). The nSeq fluid control and sample I / O coupling module 1490 controls sample and flow of samples in the nSeq biochip as well as controlling fluid and sample flow in the nSeq biochip. For example, The buffer outlet for branch # 1 is opened while all other buffer outlets are closed, if we want to perform detection in measurement chamber # 11. As a result, the fluid flows from the sample inlet towards the branch # 1 and simultaneously with the sample guide electrode along the microfluidic channel to transfer the sample to the measurement chamber # 11.

B. Software Architecture

15 shows a high-level structure of software and associated hardware for genome analysis software and an operating system in one embodiment of nanopore-based nucleotide sequencing. The various logic processing modules in the depicted software architecture may be implemented by a processing device (such as portable computing device 1410 in FIG. 14) that executes instructions embedded in a computer readable medium. The computer-readable medium provides (e.g., stores and / or stores) information in a form accessible by a computer (e.g., a server, a personal computer, a network device, a PDA, a manufacturing facility, a device having a set of one or more processors, Transmission). For example, computer readable media include recordable / non-recordable media (e.g., ROM; RAM; magnetic disk storage media; optical storage media; flash memory devices;

As noted above, the operating system 1510 is installed in the computing, communication, and control unit. The operating system 1510 may include any suitable operating system for a Windows, Linux, or computing device. Apart from the operating system 1510 installed in the computing, communication and control unit, the genome analysis software includes five processing modules: a graphical user interface (GUI) 1520, a data viewer 1530, A genomic data analyzer interface 1540, a genomic data analyzer 1550, and a genomic database 1560. Several embodiments of the interaction between the operating system 1510, the above-described processing modules 1520-1560, and other hardware components are described below with reference to FIG.

In some embodiments, the genomic data analyzer interface 1540 acts as a data flow control unit in the genome analysis software architecture. After obtaining the command and / or input data from the input device, the operating system 1510 transmits the information to the genomic data analyzer 1550 via the genomic data analyzer interface 1540. Thus, genomic data analyzer 1550 then acts. Genomic data analyzer interface 1540 controls the flow of data between I / O port 1570 and genomic database 1560, and data stored in database 1560 is stored in the database 1560, in accordance with the appropriate instructions (e.g., GET, ADD, etc.) It can be sent out or updated. Likewise, analyzer software may be periodically updated via I / O port 1570 and / or input device 1580. The genomic data analyzer interface 1540 is also coupled to the nSeq biochip interface 1590 to monitor the nSeq biochip. The status of the nSeq biochip is monitored and displayed on a display unit (such as the display device of portable computing system 1410 in FIG. 14) via an analyzer interface 1540. Genomic data analyzer interface 1540 also takes results from genomic data analyzer 1550 and indicates them to the display unit.

In some embodiments, the genomic data analyzer 1550 is the main data analysis unit of the genomic analysis software. Which obtains measurements from the nSeq biochip, performs the analysis, and then compares the results with data stored in the database 1560 to identify the bio-agents. The analysis results can be viewed in the display unit and stored in the database 1560 for future reference.

Genome database 1560 is a data repository for storing existing bio-drugs and newly discovered nucleotide sequences. Data observer 1530 includes software routines that take data and information from some or all of the other units and display them on a display device.

Thus, methods and apparatus for portable real-time analysis and identification of molecules have been described. It will be apparent from the foregoing description that aspects of the invention may be embodied, at least in part, in software. That is, the techniques may be performed in a computer system or other data processing system in response to the processor executing the sequence of instructions contained in the memory. In various embodiments, the circuitry embedded in the hardware may be used in combination with software instructions to implement the present invention. Thus, the techniques are not limited to any particular combination of hardware circuitry and software or any specific source for instructions executed by the data processing system. Moreover, all such descriptions, various functions, and operations may be described as performed or caused by the software code to simplify the description. However, those skilled in the art will recognize what this expression means by being a function resulting from the execution of the code by the processor or controller.

It is evident that reference throughout this specification to " one embodiment "or " an embodiment" means that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, it should be emphasized and understood that more than one "several embodiments" or "one embodiment" or "another embodiment" in various portions of the disclosure need not be mentioned all over the same embodiment. While the invention has been described with respect to various embodiments, it will be apparent to those skilled in the art that the present invention is not limited to the described embodiments, but, on the contrary, It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. But rather as an explanation.

Claims (21)

A first set of at least one sample guide electrode for guiding the sample towards the measurement chamber;
A second set of at least one sample guide electrode for guiding the sample away from the measurement chamber;
A nanopore array wafer having a plurality of nanopores;
An upper wafer bonded to an upper side of the nanopore array wafer;
A bottom wafer bonded to the bottom side of the nanopore array wafer;
At least one nanofluidic channel defined by a top wafer and a nanopore array wafer;
One or more microfluidic channels defined by a bottom wafer and a nanopore array wafer; And
And a plurality of measurement chambers,
Characterized in that each of the plurality of measurement chambers comprises a nano-pore, an upper driving electrode, and a bottom driving electrode.
The method according to claim 1,
Wherein each of the plurality of nanopores is fixed and has the same size.
The method according to claim 1,
A first set of sensing and biasing circuits fabricated on a top wafer, the sensing and biasing circuit comprising: a first set of sensing and biasing circuits, each of which is electrically coupled to a first set of sensing and biasing circuits, Singing circuit; And
A second set of sensing and biasing circuits fabricated on a bottom wafer, wherein a bottom driving electrode is electrically coupled to a second set of sensing and biasing circuits in each of a plurality of measurement chambers, Wherein the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of SEQ ID NO.
The method according to claim 1,
Wherein the second upper driving electrode and the second bottom driving electrode are embedded in each of the plurality of nanofores.
The method according to claim 1,
And a plurality of sensing electrodes embedded in the nano-pore array wafer, wherein each of the plurality of sensing electrodes is adjacent to one of the plurality of nano-pores.
6. The method of claim 5,
Wherein each of the plurality of sensing electrodes senses a signal selected from the group consisting of a resistance change, a capacitance change, a phase change, and a tunneling current.
The method according to claim 1,
Wherein each of the plurality of nanopores is a bio-nanopore.
8. The method of claim 7,
Wherein the bio-nanopore is a protein pore.
The method according to claim 1,
A nanopore-based nucleotide sequencing chip characterized in that the nanopore is a solid nanopore.
The method according to claim 1,
Characterized in that each of the plurality of measurement chambers performs independent analysis.
A nanopore array wafer having two layers of conductive material sandwiching a layer of dielectric material and a plurality of nanopores cut across the thickness of the nanopore array wafer;
An upper wafer bonded to an upper side of the nanopore array wafer;
A bottom wafer bonded to the bottom side of the nanopore array wafer;
At least one nanofluidic channel defined by a top wafer and a nanopore array wafer; And
At least one microfluidic channel defined by a bottom wafer and a nanopore array wafer,
Wherein an AC sensing current is applied across two layers of the conductive material to interrogate molecules traveling through the plurality of nanopores.
12. The method of claim 11,
Wherein each of the plurality of nanopores is fixed and has the same size.
12. The method of claim 11,
An upper driving electrode coupled to the bottom side of the upper wafer;
And a bottom driving electrode coupled to an upper side of the bottom wafer,
Wherein the upper driving electrode is electrically coupled to the first set of sensing and biasing circuits and the bottom driving electrode is electrically coupled to the second set of sensing and biasing circuits.
12. The method of claim 11,
Wherein the nanopore array wafer further comprises a pair of driving electrodes.
A method for producing a nanopore-based nucleotide sequencing chip usable in a portable molecular analyzer,
Depositing a layer of a first conductive material on the film layer to form a bottom conductive layer;
Depositing a dielectric material on the bottom conductive layer to form a dielectric spacer;
Depositing a layer of a second conductive material on the dielectric spacers to form an upper conductive layer;
Forming a plurality of Einzel lenses through the top conductive layer and the dielectric spacers;
Applying a nanopantographic etch on a film layer deposited on a silicon substrate of a nanopore array wafer to define a plurality of nanopores on the nanopore array wafer;
Removing a plurality of Einzel lenses after applying nanoelectrostatic etching;
Forming a protective layer on the membrane layer to protect a plurality of nanopores;
A plurality of nano pores are formed on the back surface of the silicon substrate opposite to the front surface of the silicon substrate on which the film layer is formed by using at least one of a chemical mechanical polishing (CMP) process, a lithographic process, and a potassium hydroxide process Forming a cavity for the substrate;
Removing the protective layer to expose a plurality of nanopores after the cavities on the back surface of the silicon substrate are formed;
Bonding a nanopore array wafer to a bottom side of an upper wafer to define at least one nanofluid channel between the nanopore array wafer and the upper wafer;
Bonding a nanopore array wafer to an upper side of a bottom wafer to define one or more microfluidic channels between the nanopore array wafer and the bottom wafer, wherein each nanofluid channel is configured to measure the characteristics of the molecules via nanopores The molecules being connected to corresponding ones of the microfluidic channels via corresponding nanopores so that they can migrate from the nanofluid channel to the microfluidic channel; And
Fabricating a set of sensing and biasing circuits and an upper driving electrode on the upper wafer prior to bonding the upper wafer to the nanopore array wafer,
Wherein applying nanotransfer etch comprises applying a voltage on a bottom conductive layer on which a plurality of nanometer shapes are defined such that a plurality of nanopores are formed where a plurality of nanometer shapes are defined, A method for producing a nanopore-based sequencing chip usable in an available molecular analyzer.
A method for producing a nanopore-based nucleotide sequencing chip usable in a portable molecular analyzer,
Applying nanotransfer etching on a film layer of a conductive material deposited on a silicon substrate of a nanopore array wafer to define a plurality of nanopores on the nanopore array wafer, wherein the film layer is arranged along each nanopore And a composite film having at least two conductive electrodes sandwiched between layers of dielectric material;
Bonding a nanopore array wafer to a bottom side of an upper wafer to define at least one nanofluid channel between the nanopore array wafer and the upper wafer;
Bonding a nanopore array wafer to an upper side of a bottom wafer to define one or more microfluidic channels between the nanopore array wafer and the bottom wafer, wherein each nanofluid channel is configured to measure the characteristics of the molecules via nanopores The molecules being connected to corresponding ones of the microfluidic channels via corresponding nanopores so that they can migrate from the nanofluid channel to the microfluidic channel; And
And a step of preparing a set of sensing and biasing circuits and an upper driving electrode on the upper wafer before bonding the upper wafer to the nano-pore array wafer, characterized in that the nanopore- A method for manufacturing a crystal chip.
A method for producing a nanopore-based nucleotide sequencing chip usable in a portable molecular analyzer,
Applying a nano-transfer method etch on a layer of conductive material deposited on a silicon substrate of a nanopore array wafer to define a plurality of nanopores on a nanopore array wafer, The composite film having two or more conductive electrodes arranged and sandwiched between two layers of dielectric material;
Bonding a nanopore array wafer to a bottom side of an upper wafer to define at least one nanofluid channel between the nanopore array wafer and the upper wafer;
Bonding a nanopore array wafer to an upper side of a bottom wafer to define one or more microfluidic channels between the nanopore array wafer and the bottom wafer, wherein each nanofluid channel is configured to measure the characteristics of the molecules via nanopores The molecules being connected to corresponding ones of the microfluidic channels via corresponding nanopores so that they can migrate from the nanofluid channel to the microfluidic channel; And
And a step of preparing a set of sensing and biasing circuits and an upper driving electrode on the upper wafer before bonding the upper wafer to the nano-pore array wafer, characterized in that the nanopore- A method for manufacturing a crystal chip.
18. The method according to any one of claims 15 to 17,
Wherein each of the plurality of nanopores is fixed and has the same size. 2. The method of claim 1, wherein the plurality of nanopores are fixed.
18. The method according to any one of claims 15 to 17,
Wherein the size of each nanopore in the plurality of nanopores is in the range of 1 nm to 200 nm.
18. The method according to any one of claims 15 to 17,
Wherein the plurality of nanopores are circular, elliptical, or slit-shaped. ≪ RTI ID = 0.0 > 11. < / RTI >
18. The method according to any one of claims 15 to 17,
A collimated monoenergetic ion beam collimated into an array of submicron-diameter electrostatic lenses so as to define a number of nanometer features on the film layer on the silicon substrate of the nanopore array wafer. Wherein the nanopore-based nucleotide sequencing chip comprises a nucleotide sequence of SEQ ID NO.

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